Lipid membrane structure including bacterial cell component having dispersibility in non-polar solvent, and method for producing same

ABSTRACT

[Problem] To provide: a lipid membrane structure which has such a particle diameter that the lipid membrane structure can be sterilized by filtration, contains a lipid that is bound to a peptide composed of multiple arginine residues as a constituent lipid, and includes a bacterial cell component having dispersibility in a non-polar solvent; and a method for producing a lipid membrane structure which has such a particle diameter that the lipid membrane structure can be sterilized by filtration and includes a substance of interest having dispersibility in a non-polar solvent. 
     [Solution] A lipid membrane structure which has such a particle diameter that the lipid membrane structure can be sterilized by filtration, contains a lipid that is bound to a peptide composed of multiple arginine residues as a constituent lipid, and includes a bacterial cell component having dispersibility in a non-polar solvent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application PCT/JP2013/072885, filed Aug. 27, 2013 whichclaims priority to Japanese Application No. 2012-188129, filed Aug. 28,2012, the contents of which are incorporated herein by reference intheir entireties for all purposes.

TECHNICAL FIELD

The present invention relates to a lipid membrane structureencapsulating a bacterial cell component having dispersibility in anon-polar solvent, and a method for producing the same. Morespecifically, the present invention relates to a lipid membranestructure having a particle size that permits filtration sterilization,comprising a lipid bound with a peptide consisting of a plurality ofarginine residues as a constituent lipid and encapsulating a bacterialcell component having dispersibility in a non-polar solvent, and amethod for producing a lipid membrane structure having a particle sizethat permits filtration sterilization and encapsulating a substance ofinterest having dispersibility in a non-polar solvent.

BACKGROUND OF THE INVENTION

Various carriers have been developed in order to deliver substances ofinterest such as drugs to cells. Particularly, when the substances ofinterest are bacterial cell components such as a cell-wall skeleton(CWS) fraction of Mycobacterium Bevis bacillus Calmette-Guerin (BCG),strong immunostimulatory activity is expected. These bacterial cellcomponents, however, are insoluble in water and are therefore difficultto formulate into forms applicable to organisms. Another problem of thebacterial cell components is low interaction with cells due to theirlarge molecular weights and negative chargeability. Accordingly,carriers containing surfactants, lipid membrane structures, and the likehave been proposed as carriers for the bacterial cell components. Forexample, a preparation for cancer immunotherapy comprising a bacterialcell component, an oil substance, a surfactant, and a stabilizer (PatentLiterature 1) has been disclosed. In addition, the present inventorshave disclosed a liposome comprising a bacterial cell component in alipid membrane (Patent Literature 2).

CITATION LIST Patent Literature

Patent Literature 1: International Publication No. WO2000/03724

Patent Literature 2: International Publication No. WO2007/132790

SUMMARY OF THE INVENTION Technical Problem

From the description of Patent Literature 1, however, it is not clearwhether or not the preparation for cancer immunotherapy described inPatent Literature 1 encapsulates a bacterial cell component. If thepreparation is interpreted as encapsulating a bacterial cell component,its particle size as large as a size exceeding at least 2 μm makesfiltration sterilization difficult. The liposome described in PatentLiterature 2 has a lipid membrane comprising a bacterial cell component,i.e., comprises the bacterial cell component integrally with the lipidmembrane. The liposome, however, is not a lipid membrane structureencapsulating the bacterial cell component. Although Patent Literatures1 and 2 describe the production of an oil-in-water emulsion preparation,the production method has no step of preparing a polar solvent solutioncontaining an empty lipid membrane structure. As a result, a lipidmembrane structure having a particle size that permits filtrationsterilization and encapsulating a substance of interest havingdispersibility in a non-polar solvent has not vet been produced.

The present invention has been made to solve such problems, and anobject of the present invention is to provide a lipid membrane structurehaving a particle size that permits filtration sterilization, comprisinga lipid bound with a peptide consisting of a plurality of arginineresidues as a constituent lipid and encapsulating a bacterial cellcomponent having dispersibility in a non-polar solvent, and a method forproducing a lipid membrane structure having a particle size that permitsfiltration sterilization and encapsulating a substance of interesthaving dispersibility in a non-polar solvent.

Solution to Problem

The present inventors have conducted diligent studies and consequentlycompleted the present invention by finding that: a lipid membranestructure having a particle size that permits filtration sterilizationand encapsulating a substance of interest having dispersibility in anon-polar solvent can be produced by mixing a polar solvent solutioncontaining a lipid membrane structure encapsulating no substance ofinterest with a non-polar solvent solution in which the substance ofinterest dispersed to prepare an oil-in-water emulsion, and thendistilling off the non-polar solvent under reduced pressure; and thethus-produced lipid membrane structure which has a particle size thatpermits filtration sterilization, comprises a lipid bound with a peptideconsisting of a plurality of arginine residues as a constituent lipid,and encapsulates a bacterial cell component having dispersibility in anon-polar solvent can attain the treatment of bladder cancer or theinhibition of the progression thereof and activate cellular immunity.

(1) A lipid membrane structure having a particle size that permitsfiltration sterilization, comprising a lipid bound with a peptideconsisting of a plurality of arginine residues as a constituent lipidand encapsulating a bacterial cell component having dispersibility in anon-polar solvent.

(2) The lipid membrane structure according to (1), wherein the bacterialcell component is cell-wall fraction (CW) or cell-wall skeleton fraction(CWS) of one or more bacteria selected from the group consisting of abacterium of the genus Mycobacterium, a bacterium of the genus Nocardia,a bacterium of the genus Corynebacterium, and a bacterium of the genusRhodococcus.

(3) The lipid membrane structure according to (2), wherein the bacteriumof the genus Mycobacterium is Mycobacterium Bovis bacillusCalmette-Guerin (BCG).

(4) The lipid membrane structure according to any of (1) to (3), whereinthe lipid membrane structure having a particle size that permitsfiltration sterilization is a lipid membrane structure having a particlesize of 180 nm or smaller.

(5) A pharmaceutical composition comprising a lipid membrane structureaccording to any of (1) to (4).

(6) The pharmaceutical composition according to (5), wherein thepharmaceutical composition is a therapeutic agent for bladder cancerand/or an agent inhibiting the progression thereof.

(7) A method for producing a lipid membrane structure having a particlesize that permits filtration sterilization and encapsulating a substanceof interest having dispersibility in a non-polar solvent, comprising thefollowing steps (i) to (iv):

-   -   (i) preparing a polar solvent solution containing a lipid        membrane structure encapsulating no substance of interest;    -   (ii) preparing a non-polar solvent solution in which the        substance of interest dispersed;    -   (iii) mixing the polar solvent solution containing a lipid        membrane structure encapsulating no substance of interest with        the non-polar solvent solution in which the substance of        interest dispersed to prepare an oil-in-water emulsion; and    -   (iv) distilling off the non-polar solvent under reduced pressure        from the oil-in-water emulsion.

(8) The method according to (7), wherein the lipid membrane structureencapsulating no substance of interest is a lipid membrane structurecomprising a lipid bound with a peptide consisting of a plurality ofarginine residues as a constituent lipid and encapsulating no substanceof interest.

(9) The method according to (7) or (8), wherein the substance ofinterest is a bacterial cell component.

(10) The method according to (9), wherein the bacterial cell componentis cell-wall fraction (CW) or cell-wall skeleton fraction (CWS) of oneor more bacteria selected from the group consisting of a bacterium ofthe genus Mycobacterium, a bacterium of the genus Nocardia, a bacteriumof the genus Corynebacterium, and a bacterium of the genus Rhodococcus.

(11) The method according to (10), wherein the bacterium of the genusMycobacterium is Mycobacterium Bovis bacillus Calmette-Guerin (BCG).

(12) The method according to any of (7) to (11), wherein the particlesize that permits filtration sterilization is a particle size of 180 nmor smaller.

Advantageous Effects of Invention

According to the lipid membrane structure according to the presentinvention and the method for producing the same, a lipid membranestructure that encapsulates a bacterial cell component, can be easilyformulated through dispersion in an aqueous solution, and is efficientlytaken up into cells can be obtained. A lipid membrane structureencapsulating a compactly folded particle form (particle size:approximately 100 nm) of a Mycobacterium Bovis bacillus Calmette-Guerincell-wall skeleton (BCG-CWS) fraction having a large conformation ofapproximately 1 μm in diameter in a polar solvent can be obtained.Further, a lipid membrane structure having a particle size that permitsfiltration sterilization can be obtained. Hence, the lipid membranestructure according to the present invention eliminates the need ofsterilization in the overall production process thereof forpharmaceutical use and can thus be pharmaceutically producedinexpensively by filtration sterilization. Furthermore, the lipidmembrane structure according to the present invention has a remarkablysmall content of a solvent and mainly encapsulates a substance ofinterest such as a bacterial cell component. Hence, the obtained lipidmembrane structure can efficiently deliver the substance of interest. Inaddition, the lipid membrane structure can be efficiently taken up intocancer cells to attain the treatment of cancer or the inhibition at ofthe progression thereof. Particularly, the lipid membrane structure canbe intravesically administered to an organism to attain the treatment ofbladder cancer or the inhibition of the progression thereof. Further,the lipid membrane structure can act on immunocytes to activate cellularimmunity. Furthermore, the method for producing the lipid membranestructure according to the present invention can allow the lipidmembrane structure to encapsulate the great majority of the substance ofinterest used in production so that the lipid membrane structure canefficiently encapsulate the substance of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram schematically showing a method for producing thelipid membrane structure according to the present invention.

FIG. 2 is a diagram showing the appearances of HBS, ethanol, and pentanecontaining BCG-CWS.

FIG. 3 is a diagram showing the appearances of HBS containing BCG-CWSand HBS containing a liposome encapsulating BCG-CWS.

FIG. 4 is a schematic diagram showing one embodiment of a method forpreparing a liposome encapsulating BCG-CWS.

FIG. 5 is a schematic diagram showing a method for preparing a liposomecontaining BCG-CWS in a lipid membrane (conventional form).

FIG. 6 is a diagram showing the particle size, PDI, zeta potential, andrate of encapsulation or content of BCG-CWS of each liposome comprisingR8-bound lipid as a constituent lipid at a content of 0 to 5% andencapsulating BCG-CWS.

FIG. 7 shows observation images of a liposome encapsulating BCG-CWS anda liposome encapsulating no BCG-CWS under a transmission electronmicroscopy.

FIG. 8 is a diagram showing internal solution-derived fluorescenceintensity (RFI/nmol) per nmol of lipid in a large-diameter emptyliposome, a small-diameter empty liposome, and a liposome encapsulatingBCG-CWS.

FIG. 9 is a diagram showing the positions of each liposome (green) andan acidic compartment (red) in MBT-2 cells in which an NBD-labeledliposome containing BCG-CWS in a lipid membrane or an NBD-labeledliposome encapsulating BCG-CWS was taken up. In the diagram, the arrowsdepict main sites where yellow fluorescence that indicates the overlapbetween green and red was observed.

FIG. 10 is a diagram showing the overall fluorescence intensity of eachgroup (left diagram) and the relationship between the fluorescenceintensity of each cell and the number of cells (right diagram) in anMBT-2 cell group in which an NBD-labeled liposome encapsulating BCG-CWSwas taken no (sample group) and an MBT-2 cell group in which no liposomewas taken un (control group).

FIG. 11 is a diagram showing the tumor volumes of mice that receivedtransplants of MBT-2 cells alone (group A) and mice that receivedtransplants of MBT-2 cells as well as each administered liposome (groupsB to F).

FIG. 12 is a diagram showing the number of surviving mice among micethat received transplants of MBT-2 cells as well as an administeredliposome encapsulating BCG-CWS or a administered empty liposome.

FIG. 13 is a diagram showing the total area of tumor tissues, the areaof viable tumor tissues, the area of dead tumor tissues, and the ratioof dead cells (%) in upper bar graphs wherein a represents a mouse agiven subcutaneous injection of MBT-2 cells mixed with an emptyliposome; b represents a mouse b given subcutaneous injection of MBT-2cells mixed with a liposome encapsulating BCG-CWS; c represents a mousec given subcutaneous injection of MBT-2 cells in which a liposomeencapsulating BCG-CWS was taken up; and d represents a mouse d givensubcutaneous injection of MBT-2 cells and a liposome encapsulatingBCG-CWS at sites distant from each other. The lower left diagrams arephotographs showing hematoxylin-eosin-stained tumor tissue sections ofthe mice a to d. The lower right diagrams are diagrams showing viabletumor tissues and dead tumor tissues in the hematoxylin-eosin stainimages shown in the left diagrams.

The upper diagram is a diagram showing the numbers and ratios of variousleukocytes in the tumor tissues of the mice a to d. The lower diagramsare photographs showing Giemsa-stained tumor tissue sections of the micea to d.

FIG. 15 is a diagram showing bladder sections of non-administeredbladder cancer rat models (group G) and bladder cancer rat models thatreceived each intravesically administered liposome (groups H to J).

FIG. 16 is a diagram showing the bladder weights, the number ofepithelial lesions (tumors) per 10 cm of the bladder basement membrane,and tumor volumes of non-administered bladder cancer rat models (groupG) and bladder cancer rat models chat received each intravesicallyadministered liposome (groups H to L).

FIG. 17 is a diagram showing the relationship between the production ofIFN-γ and IL-4 and the number of cells for non-supplemented naiveCD4-positive T cells (reference group) and naive CD4-positive T cellssupplemented with BCG-CW (positive control group), an empty liposome(negative control group), or a liposome encapsulating BCG-CWS (testgroup).

FIG. 18 is a diagram showing the rates of conversion (%) intoIFN-γ-producing cells and IL-4-producing cells for naive CD4-positive Tcells supplemented with BCG-CW (positive control group), a liposomeencapsulating BCG-CWS (test group), or an empty liposome (negativecontrol group).

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the lipid membrane structure encapsulating a bacterial cellcomponent having dispersibility in a non-polar solvent according to thepresent invention and the method for producing the same will bedescribed in detail. The lipid membrane structure according to thepresent invention has a particle size that permits filtrationsterilization, comprises as lipid bound with a peptide consisting of asplurality of arginine residues as a constituent lipid, and encapsulatesas bacterial cell component having dispersibility in a non-polarsolvent.

The “filtration sterilization” refers to the operation of removingmicrobes including yeasts and bacteria by filtration. The filtrationsterilization of the lipid membrane structure can be carried out, forexample, through a generally commercially available membrane filter forfiltration sterilization having a pore size of 0.2, 0.22, 0.45, or 5.0μm, or the like. Examples of the “particle size that permits filtrationsterilization” according to the present invention can preferably includeparticle sizes of 150 nm or larger and smaller than 200 nm and can morepreferably include particle sizes of 155 nm or larger and 195 nm orsmaller, further preferably 190, 187, 185, 183, 181, 180, 179, 178, 177,176, 175, 174, 173, 172, 171, 170, 169, 168, 167, 166, 165, 164, 163,162, 161, and 160 nm.

In a preferred embodiment, the lipid membrane structure according to thepresent invention is a closed vesicle having lipid membrane(s) composedof a lipid monolayer or a lipid bilayer. In this context, since thelipid membrane structure according to the present invention encapsulatesa substance of interest such as a bacterial cell component havingdispersibility in a non-polar solvent, the innermost lipid membrane mayform a lipid membrane of a lipid monolayer in which lipid molecules arearranged with their hydrophobic groups facing the lumen (substance ofinterest).

The number of lipid membrane(s) carried by the lipid membrane structureaccording to the present invention may be 1 or may be 2 or more.Specific examples of the lipid membrane structure can include amultilamellar liposome vesicle (MLV) having a plurality of lipidmembranes each composed of a lipid bilayer as well as unilamellarliposomes having only one lipid membrane composed of a lipid bilayer,such as a small unilamellar vesicle (SUV), a large unilamellar vesicle(LUV), and a giant unilamellar vesicle (GUV).

The lipid constituting the lipid membrane structure according to thepresent invention (hereinafter, this lipid is referred to as a“constituent lipid”) is not limited by its type. Examples of the lipidcan include phospholipid glycolipids, sterols, long-chain aliphaticalcohols, and glycerin fatty acid esters, any of which can be used.Also, any of cationic lipids, pH-dependent cationic lipids, neutrallipids, and anionic lipids can be used.

Examples of the phospholipids can include phosphatidylcholines (e.g.,dioleoylphosphatidylcholine, dilauroylphosphatidylcholine,dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine, and egg-yolk phosphatidylcholine (EPC)),phosphatidylglycerols (e.g., dioleoylphosphatidylglycerol,dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, and distearoylphosphatidylglycerol),phosphatidylethanolamines (e.g., dioleoylphosphatidylethanolamine,dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine(DSPE), and dioleoylglycerophosphoethanolamine (DOPE)),phosphatidylserine, phosphatidylinositol, phosphatidic acid,cardiolipin, and their hydrogenation products, and natural lipidsderived from egg yolk, soybean, or other animals or plants (e.g.,egg-yolk lecithin and soybean lecithin). One or more of thesephospholipids can be used.

Examples of the glycolipids can include: sphingomyelins;glyceroglycolipids such as sulfoxyribosyl glyceride, diglycosyldiglyceride, digalactosyl diglyceride, galactosyl diglyceride, andglycosyl diglyceride; and sphingoglycolipids such as galactosylcerebroside, lactosyl cerebroside, and ganglioside. One or more of theseglycolipids can be used.

Examples of the sterols can include: animal-derived sterols such ascholesterol (Chol), cholesterol succinate, lanosterol,dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derivedsterols (phytosterols) such as stigmasterol, sitosterol, campesterol,and brassicasterol; and microbe-derived sterols such as zymosterol andergosterol. One or more of these sterols can be used. These sterols cangenerally be used for physically or chemically stabilizing lipidbilayers or for adjusting membrane fluidity.

A fatty acid having 10 to 20 carbon atoms or an alcohol thereof can beused as a long-chain fatty acid or a long-chain aliphatic alcohol.Examples of such long-chain fatty acids or long-chain aliphatic alcoholscan include: saturated fatty acids such as palmitic acid, stearic acid(STR), lauric acid, myristic acid, pentadecylic acid, arachidic acid,margaric acid, and tuberculostearic acid; unsaturated fatty acids suchas palmitoleic acid, oleic acid, arachidonic acid, vaccenic acid,linoleic acid, linolenic acid, arachidonic acid and eleostearic acid;and oleyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol,cetyl alcohol, and linoleyl alcohol. Specific examples thereof caninclude 1,2-dimyristoyl-sn-glycerol (DMG) and 1,2-distearoyl-sn-glycerol(DSG). One or more of these long-chain fatty acids or long-chainaliphatic alcohols can be used.

Examples of the glycerin fatty acid esters can include monoacylglyceride, diacyl glyceride, and triacyl glyceride. One or more of theseglycerin fatty acid esters can be used.

Examples of the cationic lipids can include the lipids mentioned aboveas well as diethanolamine hydrochloride such as diethanolamine chloride(DC-6-14), cholesteryl hexadecyl ether (CHE),dioctadecyldimethylammonium chloride (DODAC),N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium (DOTMA),didodecylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammoniopropane (DOTAP), 3β-N-(N′,N′,-dimethyl-aminoethane)-carbamol cholesterol(DC-Chol), 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium(DMRIE), and2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminumtrifluoroacetate (DOSPA). One or more of these cationic lipids can beused.

Examples of the pH-dependent cationic lipids can include1-methyl-4,4-bis[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]piperidine (YSK05)and 1,2-dioleoyl-3-dimethylammonium propane (DODAP). One or more ofthese pH-dependent cationic lipids can be used.

Examples of the neutral lipids can include the lipids mentioned above aswell as diacylphosphatidylcholine, diacylphosphatidylethanolamine,ceramide, and dimyristoylglycerol (C14). One or more of these neutrallipids can be used. Examples of the anionic lipids can include thelipids mentioned above as well as diacylphosphatidylserine,diacylphosphatidic acid, and N-succinylphosphatidylethanolamine(N-succinyl PE), phosphatidyl ethylene glycol. One or more of theseanionic lipids can be used.

The number of arginine residues in the peptide consisting of a pluralityof arginine residues according to the present invention (hereinafter,referred to as a “peptide according to the present invention”) is notparticularly limited as long as it is two or more. Examples of thenumber of arginine residues can include several, a dozen, and severaldozens and can more specifically include 2 to 20, preferably 3 to 18,more preferably 4 to 15, further preferably 5, 6, 7, 8, 9, 10, 11, 12,13, 14, and 15. The peptide according to the present invention can besynthesized using a method appropriately selectable by those skilled inthe art on the basis of the sequence thereof. Examples of such methodscan include: a peptide synthesis method which involves chemicallypolymerizing amino acids one by one to synthesize a polypeptide; amethod which involves preparing recombinant vectors containing DNAencoding the peptide according to the present invention, transferringthe prepared vectors into appropriate host cells, and culturing theresulting transformants in a medium, followed by collection from theobtained cultures; and a method which involves expressing DNA encodingthe peptide according to the present invention in a cell-free proteinsynthesis system. General methods widely known to those skilled in theart or any of other methods can be used in these synthesis methods.

The “lipid bound with a peptide consisting of a plurality of arginineresidues” according to the present invention may be a lipid bound withthe N terminus of the peptide according to the present invention or maybe a lipid bound with the C terminus of the peptide according to thepresent invention. In the present invention, the peptide according tothe present invention and the lipid may be connected directly or may beconnected via some linker such as an amino acid (e.g., proline,isoleucine, leucine, or valine), a peptide (e.g., polyproline), or ahydrophilic polymer (e.g., polyethylene glycol (PEG)).

Examples of the binding pattern between the peptide according to thepresent invention and the lipid can include: noncovalent bonds such ashydrogen bonds, ionic bonds, hydrophobic bonds, and van der Waals bonds;and covalent bonds such as disulfide bonds and peptide bonds. Examplesof the lipid in the “lipid bound with a peptide consisting of aplurality of arginine residues” can include those listed above for useas the constituent lipid of the lipid membrane structure according tothe present invention. In Examples mentioned later, stearic acid boundwith a peptide (SEQ ID NO: 1) consisting of 8 arginine residues(stearylated octaarginine: STR-R8) was used as a “peptide-bound lipid”.

The lipid membrane of the lipid membrane structure according to thepresent invention may comprise plural types of “lipids each bound with apeptide consisting of a plurality of arginine residues”. For example,STR-R8 and palmitic acid bound with a peptide consisting of 8 arginineresidues, phospholipid bound with a peptide consisting of 10 arginineresidues and sterol bound with a peptide consisting of 7 arginineresidues, or stearic acid bound with a peptide consisting of 10 arginineresidues and glycolipid bound with a peptide consisting a 8 arginineresidues can be arbitrarily selected. Also, the content of one “lipidbound with a peptide consisting of a plurality of arginine residues” canbe appropriately determined according to the number of arginineresidues, the type of the peptide-bound lipid, the types of otherconstituent lipids, the type of the bacterial cell component, etc.

The lipid membrane of the lipid membrane structure according to thepresent invention may contain an antioxidant such as tocopherol, propylgallate, ascorbyl palmitate, or butylated hydroxytoluene, a chargedsubstance that confers positive charge, such as stearylamine oroleylamine, a charged substance that confers negative charge, such asdicetyl phosphate, a membrane protein such as membrane extrinsic proteinor membrane intrinsic protein, and the like, in addition to theconstituent lipid. The content thereof can be appropriately adjusted.

The “non-polar solvent” refers to a solvent having no polarity or asolvent having relatively small polarity. The non-polar solventaccording to the present invention is preferably a non-polar solventhaving a lower boiling point than that of a polar solvent such as water,more preferably a non-polar solvent in which the bacterial cellcomponent can be dispersed to form relatively small particles. Specificexamples of the non-polar solvent according to the present invention caninclude pentane, diethyl ether, diisopropyl ether (isopropyl ether),hexane, tetrachloromethane, toluene, benzene, dichloromethane,chloroform, cyclohexane, and butyl acetate, and mixtures thereof withacetic acid ester, acetone, or methanol. One or more of these non-polarsolvents can be used. Pentane, diethyl ether, or diisopropyl ether ispreferred.

The “bacterial cell component” according to the present invention is notparticularly limited as long as the bacterial cell component hasdispersibility in a non-polar solvent. In this context, the phrase“having dispersibility in a non-polar solvent” according to the presentinvention refers to being dispersible relatively uniformly in thenon-polar solvent. Examples of the bacterial cell component according tothe present invention can include cell-wall fraction (CW) or cell-wallskeleton fraction (CWS) of bacteria such as a bacterium of the genusMycobacterium, a bacterium of the genus Nocardia, a bacterium of thegenus Corynebacterium, and a bacterium of the genus Rhodococcus.

The cell wall (CW) of the bacterium of the genus Mycobacterium containsmacromolecules consisting of my colic acid (fatty acid), arabinogalactan(polysaccharide), and peptidoglycan, and these macromolecules constitutethe basic structure of the cell wall. This basic structure is called a.“cell wall skeleton (CWS)”. A bacterium of the genus Nocardia, abacterium of the genus Corynebacterium, a bacterium of the genusRhodococcus, and the like, which are taxonomically related to thebacterium of the genus Mycobacterium, also have a cell wall skeleton(CWS) similar to that of the bacterium of the genus Mycobacterium.

Examples of the bacterium of the genus Mycobacterium can includetuberculosis complexes such as M. tuberculosis, M. bovis, M. africanum,M. microti, M. canettii, and M. bovis BCG. Examples of the bacterium ofthe genus Nocardia can include N. asteroides, N. brasilliensis, and N.rubra. Examples of the bacterium of the genus Corynebacterium caninclude C. diphtheriae and C. ulcerans. The “bacterial cell component”according to the present invention is preferably a cell-wall fraction(BCG-CW) or cell-wall skeleton fraction (BCG-CWS) of BCG among thesebacteria.

The cell-wall fraction (CW) is not particularly limited by itscomposition, preparation method, etc., as long as the cell-wall fraction(CW) is composed mainly of a cell wall skeleton (CWS). The cell-wallfraction (CW) can be prepared, for example, by a method described inPatent Literature 2 mentioned above. The cell-wall skeleton fraction(CWS) refers to a fraction obtained by the purification of the cell wallskeleton (CWS) from the cell-wall fraction (CW). The cell-wall skeletonfraction (CWS) fraction can also be prepared by a method described inPatent Literature 2 mentioned above.

The cell-wall fraction (CW) or cell-well skeleton fraction (CWS) of thebacteria such as a bacterium of the genus Mycobacterium, a bacterium ofthe genus Nocardia, a bacterium of the genus Corynebacterium, and abacterium of the genus Rhodococcus have anticancer effect orimmunostimulatory activity (adjuvant activity or immunologicallyactivating effect). In the cell wall skeleton (CWS), the peptidoglycanmoiety and the mycolic acid moiety are important for the anticancereffect or the immunostimulatory activity. Components (e.g., lipomannanand trehalose mycolate) other than the cell wall skeleton (CWS)contained in the cell-wall fraction (CW) also contribute to theanticancer effect or the immunostimulatory activity.

The present invention also provides a pharmaceutical compositioncomprising the lipid membrane structure according to the presentinvention. The description about a constitution equivalent to orcorresponding to the aforementioned lipid membrane structureencapsulating a bacterial cell component having dispersibility in anon-polar solvent will be omitted as to the pharmaceutical compositioncomprising the lipid membrane structure according to the presentinvention. The pharmaceutical composition comprising the lipid membranestructure according to the present invention can be used inpharmaceutical application according to the effects of the bacterialcell component. When the bacterial cell component has, for example,anticancer effect or immunostimulatory activity, the pharmaceuticalcomposition comprising the lipid membrane structure of the presentinvention can be used as a therapeutic agent for cancers such as bladdercancer, pharyngeal cancer, stomach cancer, lung cancer, skin cancer,liver cancer, pancreas cancer, colon cancer, uterine cancer, andprostate cancer or an agent inhibiting the progression thereof, or as animmunostimulator (adjuvant).

Examples of the dosage form of the pharmaceutical composition caninclude dispersions of the lipid membrane structure and dried products(e.g., freeze-dried products and spray-dried products) thereof. Forexample, saline or a buffer solution such as a phosphate buffersolution, a citrate buffer solution, or an acetate buffer solution canbe used as the dispersion solvent. The dispersion may be supplementedwith additives, for example, saccharides, polyhydric alcohols,water-soluble polymers, nonionic surfactants, antioxidants, pHadjusters, and hydration promoters, and used.

The pharmaceutical composition can be used both in vivo and in vitro. Inthe case of using the pharmaceutical composition in vivo, examples ofits administration routes can include oral administration as well asparenteral administration such as intravesical administration,intravenous administration, intraperitoneal administration, subcutaneousadministration, and transnasal administration. The dose and thefrequency of administration thereof can be appropriately determinedaccording to the age, sex, and symptoms of a recipient, the type andamount of the bacterial cell component, etc.

The present invention further provides a method for producing the lipidmembrane structure encapsulating a substance of interest havingdispersibility in a non-polar solvent. The method for producing thelipid membrane structure according to the present invention isschematically shown in FIG. 1. The method for producing the lipidmembrane structure according to the present invention is a method forproducing a lipid membrane structure having a particle size that permitsfiltration sterilization and encapsulating a substance of interesthaving dispersibility in a non-polar solvent, comprising the followingsteps (i) to (iv):

-   -   (i) preparing a polar solvent solution containing a lipid        membrane structure encapsulating no substance of interest;    -   (ii) preparing a non-polar solvent solution in which the        substance of interest dispersed;    -   (iii) mixing the polar solvent solution containing a lipid        membrane structure encapsulating no substance of interest with        the non-polar solvent solution in which the substance of        interest dispersed to prepare an oil-in-water emulsion; and    -   (iv) distilling off the non-polar solvent under reduced pressure        from the oil-in-water emulsion.

The description about a constitution equivalent to or corresponding tothe aforementioned lipid membrane structure encapsulating a bacterialcell component having dispersibility in a non-polar solvent or theaforementioned pharmaceutical composition comprising the lipid membranestructure according to the present invention will be omitted as to themethod for producing the lipid membrane structure encapsulating asubstance of interest having dispersibility in a non-polar solventaccording to the present invention.

The “substance of interest” according to the present invention is notparticularly limited as long as the substance of interest hasdispersibility in a non-polar solvent. Examples of the “substance ofinterest” according to the present invention can include variousbiologically active substances such as drugs, nucleic acids, peptides,proteins, sugars, and complexes thereof. A bacterial cell component ispreferred.

The step (i) of preparing a polar solvent solution containing a lipidmembrane structure encapsulating no substance of interest may beperformed by a non-limiting method and can be carried out according to aroutine method. Examples of such methods can include methods whichinvolve preparing the lipid membrane structure encapsulating nosubstance of interest by a method known in the art, such as a hydrationmethod, a sonication method, an ethanol injection method, an etherinjection method, a reverse-phase evaporation method, a surfactantmethod, or a freezing-thawing method, using a polar solvent.Alternatively, the lipid membrane structure may be prepared according toa routine method, then recovered, and added to a desired polar solventto prepare a polar solvent solution, or the external solution of thelipid membrane structure may be replaced with a desired polar solvent bydialysis or the like to prepare a polar solvent solution. The number oflipid membrane(s) in the “lipid membrane structure encapsulating nosubstance of interest” may be 1 or may be 2 or more. Examples of theconstituent lipid of the “lipid membrane structure encapsulating nosubstance of interest” can include the lipids listed above.

In this context, the “polar solvent” refers to a solvent havingrelatively large polarity. The polar solvent according to the presentinvention is preferably a polar solvent having a higher boiling pointthan that of a non-polar solvent constituting an oil-in-water emulsionin the step (iii), because the non-polar solvent can be easily distilledoff under reduced pressure. Specific examples of the polar solventaccording to the present invention can include water, ethanol, aceticacid, acetonitrile, and acetone. One or more of these polar solvents canbe used.

Examples of the method for preparing a non-polar solvent solution inwhich the substance of interest dispersed in the step (ii) can includemethods which involve adding the substance of interest to a non-polarsolvent, followed by stirring. In this context, the non-polar solvent ispreferably a non-polar solvent that can be distilled off under reducedpressure from an oil-in-water emulsion in the step (iii), i.e., anon-polar solvent having a lower boiling point than that of the polarsolvent constituting the oil-in-water emulsion. Specific examplesthereof can include those listed above as the “non-polar solvent” forthe lipid membrane structure according to the present invention.

The step (iii) of mixing the polar solvent solution containing a lipidmembrane structure encapsulating no substance of interest with thenon-polar solvent solution in which the substance of interest dispersedto prepare an oil-in-water emulsion can be carried out according to aroutine method. Examples of such methods can include a method whichinvolves stirring the mixture using a vortex mixer and a method whichinvolves sonicating the mixture using a sonicator. These methods can beappropriately selected according to the types and amounts of the polarsolvent and the non-polar solvent.

Examples of the method for distilling off the non-polar solvent underreduced pressure from the oil-in-water emulsion in the step (iv) caninclude methods which involve applying the oil-in-water emulsion to anevaporator. In this case, the water bath temperature and rotationalspeed of the evaporator, the duration for which the oil-in-wateremulsion is applied to the evaporator, and the like can be appropriatelydetermined according to the types and amounts of the polar solvent andthe non-polar solvent, etc.

The lipid membrane structure according to the present invention and thelipid membrane structure produced by the method for producing the lipidmembrane structure according to the present invention can be dispersed,for use, in an appropriate aqueous solvent such as saline, a phosphatebuffer solution, a citrate buffer solution, or an acetate buffersolution. The dispersion may be appropriately supplemented withadditives, for example, saccharides, polyhydric alcohols, water-solublepolymers, nonionic surfactants, antioxidants, pH adjusters, andhydration promoters. The lipid membrane structure can be stored in adried state of the dispersion. In addition, the lipid membrane structurecan be orally administered and may also be parenterally administered,for example, through an intravesical, intravenous, intraperitoneal,subcutaneous, or transnasal route.

Hereinafter, the lipid membrane structure encapsulating a bacterial cellcomponent having dispersibility in a non-polar solvent according to thepresent invention and the method for producing the same will bedescribed with reference to Examples. It should be understood that thetechnical scope of the present invention is not limited by featuresshown by these Examples.

EXAMPLES Example 1 Preparation of Liposome (1) Liposome EncapsulatingBCG-CWS [1-1] Preparation of HBS Containing Empty Liposome

Stearic acid (STR-R8; Kurabo Industries Ltd.) bound with a peptide (SEQID NO: 1; hereinafter, referred to as “R8”) consisting of arginineresidues was prepared. The peptide R8 is known to impart a giveninternalization ability to liposomes (international Publication No.WO2005/032593: and Kentaro Kogure, Yakugaku Zasshi (Journal of thePharmaceutical Society of Japan), Vol. 127, No. 10, p. 1685-1691, 2007).STR-R8 has a structure in which stearic acid (STR) is bound with the Nterminus of R8. Subsequently, a liposome was prepared by the hydrationmethod using egg-yolk phosphatidylcholine (EPC), cholesterol (Chol), andSTR-R8. Specifically, these lipids were dissolved at a molar ratioEPC:Chol:STR-R8=70:30:2 in chloroform. This solution was used as a lipidchloroform solution (total lipid concentration: 10 mmol/L). The lipidchloroform solution was placed in an eggplant-shaped flask and driedunder reduced pressure using an evaporator to obtain a lipid film. Thelipid film was hydrated by the addition of 5 mmol/L HEPES-bufferedsaline (5 mmol/L HEPES and 0.9 w/v % NaCl; hereinafter, referred to as“HBS”) having a pH of 7.4, and stirred or sonicated to prepare aliposome, which was used as an empty liposome. The empty liposomeencapsulated no substance of interest and had a lumen filled with HBS.Subsequently, the empty liposome was filtered through a polycarbonatemembrane filter (Nucleopore Corp.) having a pore size of 400 nm usingMini Extruder (Avanti Polar Lipids, Inc.) to prepare HBS containing anempty liposome having a pose size of 400 nm or smaller.

[1-2] Preparation of Solution Containing BCG-CWS Dispersed in Non-PolarSolvent

A Mycobacterium Bovis bacillus Calmette-Guerin cell-wall skeleton(BCG-CWS) fraction was obtained according to the method described in theprevious report (Patent Literature 2). Subsequently, for a preliminaryexperiment, 1 mg of BCG-CWS was added to 300 μL each of HBS, ethanol,and pentane and stirred to confirm their appearances. The results areshown in FIG. 2. As shown in FIG. 2, BCG-CWS aggregated in HBS to form avisibly large particle. By contrast, BCG-CWS was dispersed in ethanoland pentane without aggregating. This demonstrated that BCG-CWS isdispersed in an organic solvent without aggregating.

Accordingly, next, 1 mg of BCG-CWS was added to 300 μL each of polarsolvents and non-polar solvents and dispersed by stirring. The particlesize and phase Doppler interferometer (PDI) of BCG-CWS in these solventswere measured using ZETASIZER Nano ZEN3600 (Malvern Instruments Ltd.).The polar solvents used were ethanol, 2-propanol, and tert-butylalcohol, and the non-polar solvents used were pentane, diethyl ether,diisopropyl ether, and hexane. The results are shown in Table 1. Asshown in Table 1, both of the particle size and PDI were remarkablysmall values for pentane, diethyl ether, diisopropyl ether, and hexaneas compared with ethanol, 2-propanol, and tert-butyl alcohol. Thisdemonstrated that BCG-CWS in a non-polar solvent has a smaller particlesize and also a smaller variation in particle size than those of BCG-CWSin a polar solvent. These results showed that a bacterial cell componentdispersed in a non-polar solvent has a small particle size and has arelatively uniform particle size. Thus, 1 mg of BCG-CWS was added to 300μL each of pentane, diethyl ether, diisopropyl ether, and hexane anddispersed, and these dispersions were each used as a non-polar solventsolution of BCG in the subsequent experiments.

TABLE 1 Solvent Particle size (nm) PDI Polar solvent Ethanol  2276 ±1702 0.516 ± 0.375 2-Propanol 2222 ± 636 0.438 ± 0.322 tert-Butylalcohol 3235 ± 841 0.359 ± 0.367 Non-Polar solvent Pentane 96 ± 1 0.106± 0.041 Diethyl ether 104.8 0.212 Diisopropyl ether 121 ± 3  0.291 ±0.225 Hexane 130 ± 5  0.141 ± 0.034

[1-3] Preparation of Oil-in-Water Emulsion and Distilling Off ofNon-Polar Solvent under Reduced Pressure

The HBS containing an empty liposome of Example 1(1)[1-1] was added inan amount of 700 μL to 300 μL of each non-polar solvent solution of BCGof Example 1(1)[1-2] and mixed therewith by sonication using aprobe-type sonicator to prepare an oil-in-water emulsion (O/W emulsion).Subsequently, the O/W emulsion was transferred to an eggplant-shapedflask and subjected to evaporation using an evaporator so that pentane,diethyl ether, diisopropyl ether, or hexane was distilled off underreduced pressure to prepare a liposome, which was used as a liposomeencapsulating BCG-CWS. Subsequently, each liposome encapsulating BCG-CWSwas filtered through a polycarbonate membrane filter (Nucleopore Corp.)having a pore size of 200 nm using Mini Extruder (Avanti Polar LipidsInc.). The appearance of HBS containing the liposome encapsulatingBCG-CWS was compared with the appearance of HBS containing BCG-CWS. Theresults are shown in FIG. 3. As mentioned above, BCG-CWS aggregated inHBS to form a visibly large particle, whereas the liposome encapsulatingBCG-CWS was confirmed to be dispersed in HBS as shown in FIG. 3. Themethod for preparing the liposome encapsulating BCG-CWS according toExamples 1(1)[1-1] to 1(1)[1-3] is schematically shown in FIG. 4.

(2) Liposome for Comparison

A liposome was prepared according to the methods described in Examples1(1)[1-1] to 1 (1)[1-3] and used as a liposome for comparison. However,pentane containing EPC, Chol, and STR-R8 at a molar ratio ofEPC:Chol:STR-R8=70:30:2 and at a total lipid concentration of 23.3mmol/L and containing BCG-CWS at a concentration of 1 mg/300 μL was usedinstead of the non-polar solvent solution of BCG, and HBS was usedinstead of the HBS containing an empty liposome.

(3) Liposomes Containing Varying Amounts of R8

Liposomes were prepared according to the method described in Example1(1)[1-1] except that the ratio of the lipids in the lipid chloroformsolution was changed to EPC:Chol:STR-R8=70:30:0, 1, 2, 3, 4, and 5instead of EPC:Chol:STR-R8=70:30:2. Specifically, empty liposomes having0, 1, 2, 3, 4, or 5% content of the R8-bound lipid as a constituentlipid with respect to the total amount of other constituent lipids wereprepared and used as R8/0 to 5% empty liposomes. Next, liposomes wereprepared according to the methods described in Examples 1(1)[1-2] and1(1)[1-3] except that pentane wee used as a non-polar solvent and theR8/0 to 5% empty liposomes were each used instead of the empty liposome.These liposomes were used as R8/0 to 5% liposomes.

(4) Liposome Containing BCG-CWS in Lipid Membrane

A liposome having a lipid membrane comprising BCG-CWS was preparedaccording to the method described in the previous report (InternationalPublication No. WO2007/132790) and used as a liposome containing BCG-CWSin a lipid membrane. Specifically, EPC and Chol were first dissolved ata molar ratio of EPC:Chol=7:3 in chloroform to obtain an EPC/Cholsolution (total lipid concentration: 10 mmol/L). Also, STR-R8 wasdissolved at a concentration of 10 mmol/L in water to obtain as aqueousSTR-R8 solution. Further, 1 mg of BCG-CWS was suspended in achloroform/ethanol mixed solvent of chloroform:ethanol=2:1 (v:v) toobtain a BCG-CWS suspension. Subsequently, the whole amount of theBCG-CWS suspension (amount of BCG-CWS: 1 mg) was mixed with the EPC/Cholsolution and the aqueous STR-R8 solution at a ratio of 100:2 (v:v) toobtain a mixed lipid solution of BCG. The mixed lipid solution of BCGwas applied to an evaporator and dried under reduced pressure to preparea lipid film. The lipid film was hydrated by the addition of 700 μL ofHBS. Then, glass beads were added thereto and stirred by the rotation ofa rotary evaporator at 65° C. for 20 minutes without reduction inpressure to prepare a liposome containing BCG-CWS in a lipid membrane.Subsequently, the liposome was filtered through membrane filters havingpore sizes of 1,000 nm and 400 nm. The method for preparing the liposomecontaining BCG-CWS in a lipid membrane according to Example 1(4) isschematically shown in FIG. 5.

Example 2 Properties of Liposome (1) Particle Size, PDI, Zeta Potential,and Rate of Encapsulation or Content of BCG-CWS [1-1] Measurement Method

The particle size, PDI, and zeta potential of each liposome weremeasured by the dynamic light scattering method for the particle sizeand PDI and by electrophoresis for the zeta potential using ZETASIZERNano ZEN3600 (Malvern Instruments Ltd.).

The rate of encapsulation or content of BCG-CWS of each liposome wasalso measured as follows: first, BCG-CWS was suspended at aconcentration of 0.05 mg/mL, 0.25 mg/mL, or 0.1 mg/mL in ethanol, andthese suspensions were each used as a sample for calibration. Also, HBScontaining each liposome was used as a sample for assay. Water andphenol were added to a 1.1% (v/v) fuchsin solution (Nacalai Tesque,Inc.) to adjust she final concentration of phenol to 5% (w/v) and thefinal concentration of fuchsin to 0.55% (v/v). This solution was used asa fuchsin/phenol solution. Next, 1 mL of 100% ethanol was placed in eachmicrotube. Further, 1 mL of 100% ethanol (negative control), 1 mL ofeach sample for calibration, or 100 to 150 μL each sample for assay wasplaced therein. These microtubes were vortexed for 2 minutes to depositBCG-CWS. The pellet of the deposited BCG-CWS was visually confirmed andthen centrifuged for 5 minutes under conditions of 15° C. and 6000 rpmto remove the supernatant and recover the pellet. Subsequently, 1 mL of100% ethanol was added thereto, and centrifugation was performed underthe same conditions as above to remove the supernatant and recover thepellet. The pellet was dried in air for 10 minutes. Then, 400 μL ofhexane was added thereto, and BCG-CWS was dispersed into hexane byvortex or sonication. Subsequently, 200 μL of the fuchsin/phenolsolution was added to the dispersion and inverted for 3 to 4 minutes tostain BCG-CWS with fuchsin. In this context, mycolic acid, which is amain component of BCG-CWS, is known to react with fuchsin to develop redcolor. Subsequently, 250 μL of the upper hexane layer was recovered andtransferred to a 96-well plate, and the absorbance was measured at 530nm. A calibration curve of BCG-CWS concentrations was prepared on thebasis of the measurement results of the negative control and the samplesfor calibration. The measurement results of the sample for assay werefit into the prepared calibration curve to determine the BCG-CWSconcentration of the sample for assay. The amount of BCG-CWS in thetotal amount of the liposome was calculated from this BCG-CWSconcentration, and the rate of encapsulation or content of BCG-CWS wasdetermined according to the following expression 1:

Rate of encapsulation or content of BCG-CWS (%)=100×(Amount of BCG-CWSin the total amount of the liposome/Amount of BCG-CWS used in thepreparation of the liposome).  Expression 1;

Specifically, the rate of encapsulation or content of BCG-CWS refers toa value indicating the ratio of BCG-CWS actually encapsulated in theliposome or contained in the lipid membrane to BCG-CWS used in thepreparation of the liposome.

[1-2] Comparison between Liposome Encapsulating BCG-CWS and LiposomeContaining BCG-CWS in Lipid Membrane

The particle size, PDI, zeta potential, and the rate of encapsulation orcontent of BCG-CWS were measured by the methods described in Example2(1)[1-1] as to the liposomes encapsulating BCG-CWS of Example 1(1) andthe liposome containing BCG-CWS in a lipid membrane of Example 1(4). Theresults are shown in Table 2. As shown in Table 2, PDI and the zetapotential were equivalent between each liposome encapsulating BCG-CWSand the liposome containing BCG-CWS in a lipid membrane. By contrast,the particle size was remarkably small in the liposome encapsulatingBCG-CWS as compared with the liposome containing BCG-CWS in a lipidmembrane. Also, the liposome encapsulating BCG-CWS has a particle sizeof 180 nm or smaller when prepared using any of pentane, diethyl ether,diisopropyl ether, and hexane. These results showed that the lipidmembrane structure prepared by the methods shown in Examples 1(1)[1-1]to 1(1)[1-3] has a particle size that permits filtration sterilization.The rate of encapsulation or content of BCG-CWS was remarkably large inthe liposome encapsulating BCG-CWS prepared using pentane or diisopropylether as compared with the liposome containing BCG-CWS in a lipidmembrane. These results revealed that the methods shown in Examples1(1)[1-1] to 1(1)[1-3] can allow the lipid membrane structure toefficiently encapsulate the substance of interest.

TABLE 2 Particle size Zeta potential Rate of encapsulation (nm) PDI (mV)or content of BCG-CWS Liposome encapsulation BCG-CWS 166 ± 4 0.257 ±0.04  31.2 ± 0.8 57.0 ± 4.0 (prepared using pentane) Liposomeencapsulation BCG-CWS 168.1 0.356 (prepared using diethyl ether)Liposome encapsulation BCG-CWS 172.8 ± 5.4 0.293 ± 0.020 35.2 ± 1.0 50.5± 2.2 (prepared using diisopropyl ether) Liposome encapsulation BCG-CWS146.7 ± 5.5 0.199 ± 0.012  40.7 ± 53.2 (prepared using hexane) Liposomecontaining BCG-CWS   496 ± 1.32 0.453 ± 0.08  33.5 ± 6.0 37.5 ± 8.8 inlipid membrane

In Examples 2(1)[1-3] and 2(2) to 2(5) and Examples 3 to 5 given below,the liposome encapsulating BCG-CWS of Example 1(1)[1-3] prepared usingpentane was used.

[1-3] Comparison between Liposome Encapsulating BCG-CWS and Liposome forComparison

The particle size, PDI, zeta potential, and the rate of encapsulation orcontent of BCG-CWS were measured by the methods described in Example2(1)[1-1] as to the liposome for comparison of Example 1(2). The resultsare shown in Table 3 in comparison to the results about the liposomeencapsulating BCG-CWS of Example 1(1). As shown in Table 3, the particlesize, PDI, and the zeta potential were equivalent between the liposomeencapsulating BCG-CWS and the liposome for comparison. On the otherhand, the rate of encapsulation or content of BCG-CWS was remarkablylarge in the liposome encapsulating BCG-CWS as compared with theliposome for comparison. This revealed that for the efficientencapsulation of BCG-CWS in the liposome, it is required to mix the HBScontaining an empty liposome with the non-polar solvent solution of BCG.These results showed that for the efficient encapsulation of thesubstance of interest in the lipid membrane structure, it is required tomix the polar solvent solution containing a lipid membrane structureencapsulating no substance of interest with the non-polar solventsolution in which the substance of interest dispersed to prepare anoil-in-water emulsion.

TABLE 3 Rate of Particle Zeta encapsulation size potential or content of(nm) PDI (mV) BCG-CWS Liposome 166 ± 4  0.257 ± 0.04  31.2 ± 0.8 57.0 ±4.0  encapsulating BCG-CWS (prepared using pentane) Liposome for 157 ±18 0.210 ± 0.011 40.0 ± 3.8 12 ± 19 comparison

[1-4] Comparison among Liposomes Containing Varying Amounts of R8

The particle size, PDI, zeta potential, and the rate of encapsulation orcontent of BCG-CWS were measured by the methods described in Example2(1)[1-1] as to the R8/0 to 5% liposomes of Example 1(3). The resultsare shown in FIG. 6. As shown in FIG. 6, the particle size wassubstantially equivalent among the R8/2% to 5% liposomes, but wasparticularly small in the R8/2% liposome. These results showed that theparticle size of the lipid membrane structure is particularly small whenthe content of the lipid bound with a peptide consisting of a pluralityof arginine residues is 1% or larger and smaller than 3% with respect tothe total amount of other constituent lipids.

Next, PDI was substantially equivalent among the R8/0 to 5% liposomes,but was particularly small in the R8/0% liposome. This seemed to bebecause the R8/0% liposome encapsulated no BCG-CWS, as mentioned later.Next, the zeta potential was a negative value (anionic) in the R8/0%liposome, but was a positive value (cationic) in all of the R8/1 to 5%liposomes. This seemed to be because the R8/0% liposome was free fromthe cationic substance STR-R8.

Finally, the rate of encapsulation or content of BCG-CWS was equal to orsmaller than the detection limit (Not Detected; N.D.) in the R8/0%liposome, but was 15% or larger on average in all of the R8/1 to 5%liposomes. This means that when the lipid membrane structureencapsulating no substance of interest had no R8-bound lipid as aconstituent lipid, a lipid membrane structure having a particle sizethat permitted filtration sterilization and encapsulating a bacterialcell component was difficult to prepare; and by contrast, when the lipidmembrane structure encapsulating no substance of interest had R8-boundlipid as a constituent lipid at a given concentration, a lipid membranestructure having a particle size that permitted filtration sterilizationand encapsulating a bacterial cell component was successfully prepared.These results showed that even if the number of arginine residues in thepeptide consisting of arginine residues is changed, a lipid membranestructure having a particle size that permits filtration sterilizationand encapsulating a bacterial cell component can be prepared by changingthe content of the lipid bound with a peptide consisting of a pluralityof arginine residues and modifying the peptide.

(2) Microscopic Observation

HBS containing the liposome encapsulating BCG-CWS of Example 1(1) wasprepared. This HBS was mixed with a 2% (v/v) aqueous tungstic acidsolution and then added dropwise to a carbon vapor-deposited 400-meshgrid. Excessive water was removed, and the grid was dried in air, thenobserved at an acceleration voltage of 80 kV using a transmissionelectron microscopy JEM-1200EX (JEOL Ltd.), and photographed using a CCDcameral (Olympus Soft imaging Solutions GmbH). In this observation undera transmission electron microscopy, BCG-CWS is observed as an irregularstrand-like or sheet-like (uniform plane with almost no contrast)structure (Y. Uenishi at al., Journal of Microbiological Methods, Vol.77, p. 139-144, 2009). This is presumably because BCG-CWS assumes anonuniform structure such as a long chain structure or a foldedstructure in a solvent. On the other hand, the liposome has a uniformvesicle structure and as such is probably observed as a concentricstructure.

The photographing results are shown in FIG. 7. As shown in the leftdiagram of FIG. 7, for the liposome encapsulating BCG-CWS, irregularstrand-like and sheet-like structures (which are probably of BCG-CWS)were observed within the concentric structure (which is probably of theliposome). These irregular strand-like and sheet-like structures were ina compactly folded particle form around which a gap indicating thepresence of the solvent (pentane or HBS) was hardly observed. Thisrevealed that the liposome encapsulating BCG-CWS encapsulated BCG-CWSand hardly encapsulated the solvent. As shown in the right diagram ofFIG. 7, a liposome encapsulating no BCG-CWS was also observed in the HBScontaining the liposome encapsulating BCG-CWS. In the liposomeencapsulating no BCG-CWS, a morphological form was observed in whichlipid membranes were densely packed up to the central portion of theconcentric structure and few luminal portions were existed. Thisrevealed that the methods shown in Examples 1(1)[1-1] to 1(1)[1-3],which involve distilling off the non-polar solvent under reducedpressure from the O/W emulsion, almost completely distill off thenon-polar solvent present in the liposome lumen and produce a liposomehardly encapsulating the solvent.

These results showed that: the methods shown in Examples 1(1)[1-1] to1(1)[1-3] can produce a lipid membrane structure encapsulating asubstance of interest having dispersibility in a non-polar solvent; andthe amount of the solvent encapsulated by this lipid membrane structureis very small.

(3) Amount of Polar Solvent Contained in Liposome; Study on FluorescenceIntensity Derived from Polar Solvent Contained in Liposome

Each empty liposome was prepared by the method described in Example1(1)[1-1], while a liposome encapsulating BCG-CWS was prepared by themethods described in Examples 1(1)[1-1] to 1(1)[1-3]. However, HBSpresent inside and outside each liposome was fluorescently stained byuse of HBS containing 0.1 mmol/L calcein instead of HBS. Also, membranefilters having a pore size of 400 nm and a pore size of 200 nm were usedto filter the empty liposomes. The empty liposome filtered through theformer membrane filter was used as a large-diameter empty liposome, andthe empty liposome filtered through the latter membrane filter was usedas a small-diameter empty liposome. The particle size of each preparedliposome was measured using Zetasizer Nano ZS (Malvern Instruments Ltd.)and was consequently 248 nm for the large-diameter empty liposome, 160nm for the small-diameter empty liposome, and 161 nm for the liposomeencapsulating BCG-CWS.

Calcein is known to quench through complexation in the presence ofcobalt chloride. On the basis of this fact, cobalt chloride was firstadded to the external solution of each liposome to eliminate thefluorescence of calcein in the external solution. Subsequently, thefluorescence intensity (relative fluorescence intensity: RFI) wasmeasured at an excitation wavelength of 460 nm and a fluorescencewavelength of 550 nm using a fluorophotometer and used as fluorescenceintensity A. The fluorescence intensity A principally indicates theamount of fluorescence derived from HBS (HBS present in the lumen of theliposome and the gap in the lipid bilayer of the liposome) contained inthe liposome. Next, the lipid membrane of the liposome was disruptedusing 1% (v/v) Triton X-100 to eliminate the fluorescence of calcein inHBS contained in the liposome. Then, the fluorescence intensity wassimilarly measured and used as fluorescence intensity B. Thefluorescence intensity B indicates the amount of background fluorescenceafter quenching of calcein inside and outside the liposome. Also, thetotal lipid amount (nmol) of the solution in which the liposome wascontained was measured using a phospholipid quantification kit (WakoPure Chemical Industries, Ltd.). On the basis of the measurementresults, the fluorescence intensity truly derived from HBS contained inthe liposome (fluorescence intensity derived from contained HBS) wascalculated according to the following expression 2:

Fluorescence intensity derived from contained HBS (RFI)=Fluorescenceintensity A−Fluorescence intensity B.  Expression 2;

Next, the value of the fluorescence intensity derived from contained HBSwas divided by the total lipid amount to determine fluorescenceintensity derived from contained HBS per nmol of lipid (RFI/nmol). Thisexperiment of Example 2(3) was conducted 4 times to determine a mean ofRFI/nmol and standard deviation. RFI/nmol of the liposome encapsulatingBCG-CWS was subjected to a significance test (two-way repeated measuresANOVA, Tukey-Kramer method) vs. the large-diameter empty liposome andthe small-diameter empty liposome. The results are shown in FIG. 6.

As shown in FIG. 6, RFI/nmol of the liposome encapsulating BCG-CWS wassignificantly small as compared with the large-diameter empty liposomeand the small-diameter empty liposome. RFI/nmol of the liposomeencapsulating BCG-CWS was smaller than 3500 even in consideration of therange of the standard deviation. This revealed that the amount of HBScontained in the liposome encapsulating BCG-CWS was remarkably small ascompared with the empty liposome having a larger particle size and eventhe empty liposome having almost the same particle size. These resultsshowed that the amount of the polar solvent contained in the lipidmembrane structure prepared by the methods shown in Examples 1(1)[1-1]to 1(1)[1-3] is remarkably small. These results also showed that whenthe polar solvent contained in the lipid membrane structure is 10 mmol/LHBS containing calcein at a final concentration of 0.1 mmol/L and havinga pH of 7,4, the fluorescence intensity of calcein derived from thepolar solvent contained in the lipid membrane structure is less than3500 per nmol of the constituent lipid of the lipid membrane structurein the measurement at an excitation wavelength of 460 nm and afluorescence wavelength of 550 nm.

(4) Amount of Polar Solvent Contained in Liposome; Study Based onReduction in Fluorescence Intensity

When the liposome is disrupted by Triton X-100, the external solution ofthe liposome is diluted with the polar solvent contained in the liposometo decrease the concentration of calcein, resulting in reduction influorescence intensity. On the basis of this fact, the amount of thepolar solvent contained in each liposome was measured. Specifically, anempty liposome was prepared by the method described in Example1(1)[1-1], while a liposome encapsulating BCG-CWS was prepared by themethods described in Examples 1(1)[1-1] to 1(1)[1-3]. To 10 μL of HBScontaining each liposome, 970 μL of HBS and 20 μL of a 0.1 mmol/Lcalcein solution were added. This mixture was halved into 500 μL. One ofthe portions was supplemented with 5 μL of 10% (v/v) Triton X-100 finalconcentration: 0.1% (v/v)) and used as a solution B. The other portionwas supplemented with 5 μL of HBS and used as a solution A.Approximately 300 μL aliquots of the solution A and the solution B wereeach added to 100 kDa Microcon (EMD Millipore) and ultrafiltered bycentrifugation for 10 minutes under conditions of 5000 rpm and 4° C.From the non-ultrafiltered samples and the ultrafiltered samples, 100 μLaliquots were separated, and the fluorescence intensity was measured.

As a result, the fluorescence intensity was smaller in the solution Bthan in the solution A. The degree of reduction in fluorescenceintensity in the solution B compared with the solution A was smaller inthe liposome encapsulating BCG-CWS than in the empty liposome. Theseresults showed that the amount of the polar solvent contained in thelipid membrane structure prepared by the methods shown in Examples1(1)[1-1] to 1(1)[1-3] is remarkably small.

(5) Amount of Polar Solvent Contained in Liposome; Study Based on WeightMeasurement

A liposome encapsulating BCG-CWS was prepared by the methods describedin Examples 1(1)[1-1] to 1(1)[1-3]. Subsequently, the liposome wasfreeze-dried to obtain a freeze-dried product. The weight of thefreeze-dried product was measured. Then, the freeze-dried product wasresuspended by the addition at 1 mL of DDW to prepare a resuspendedliposome. Subsequently, the resuspended liposome was separated from theexternal aqueous layer by centrifugation for 30 minutes under conditionsof 43000 rpm and 4° C. Then, the wet weight of the resuspended liposomewas measured. Also, the lipid concentration thereof was measured using aphospholipid quantification kit (Wako Pure Chemical Industries, Ltd.).The wet weight of the resuspended liposome was corrected on the basis ofthe measurement results. Subsequently, the weight of the aqueoussolution contained in the liposome encapsulating BCG-CWS was determinedaccording to the following expression 3:

Corrected wet weight of the resuspended liposome−Weight of thefreeze-dried product.  Expression 3;

Example 3 Cellular Uptake (1) Preparation of Liposome

A liposome encapsulating BCG-CWS was prepared by the methods describedin Examples 1(1)[1-1] to 1(1)[1-3], while a liposome containing BCG-CWSin a lipid membrane was prepared by the method described in Example1(4). However, nitro-2-1,3-benzoxadiazol-4-yl (NBD)-bound DOPE(NBD-labeled DOPE; Avanti Polar Lipids, Inc.) was added at a molar ratioof 1% with respect to the total amount of other constituent lipids toeach of the lipid chloroform solution and the mixed lipid solution ofBCG to label the surface of each liposome with NBD.

(2) Fluorescent Observation

Mouse bladder cancer cell line MBT-2 cells (RIKEN, Japan) wereinoculated at a density of 2×10⁵ cells to a plate and cultured at 37° C.for 1 day in a 5% (v/v) CO₂ atmosphere using an RPMI1640 mediumsupplemented with 10% fetal calf serum (FCS) (FCS-supplemented RPMImedium). After removal of the medium, the cells were washed with 1 mL ofphosphate-buffered saline (PBS). Subsequently, 1 mL of an RPMI1640medium containing the liposome encapsulating BCG-CWS or the liposomecontaining BCG-CWS in a lipid membrane in Example 3(1) at a total lipidconcentration of 75 μmol/L was added to the cells. The liposome wastaken on into the MBT-2 cells by incubation for 1 hour under the sameconditions as above. After removal of the medium, the cells were washedtwo repetitive times with 1 mL of PBS containing 20 U/mL heparin andthen washed once with 1 mL of an FCS-supplemented RPMI medium.Subsequently, 1 mL of an FCS-supplemented RPMI medium was added to thecells, which were then incubated for 45 minutes under the sameconditions as above. Next, 5 μL of 100 μmol/L LysoTracker Red was addedto the cells, which were further incubated for 15 minutes to stain theacidic compartments of the MBT-2 cells. Then, the cells were washed tworepetitive times with 1 mL of an FCS-supplemented RPMI medium. Then, 1mL of an FCS-supplemented RPMI medium was added thereto, and thefluorescence of NBD (green) and LysoTracker Red (red) was observed undera confocal laser scanning microscope (LSM510; Carl Zeiss AG). Theresults are shown in FIG. 9.

As shown in FIG. 9, green fluorescence and yellow fluorescenceindicating the overlap between green and red were observed within theMBT-2 cells in which the liposome containing BCG-CWS in a lipid membraneor the liposome encapsulating BCG-CWS was taken up. The number of siteswhere the yellow fluorescence was observed was larger when the liposomeencapsulating BCG-CWS was taken up than when the liposome containingBCG-CWS in a lipid membrane was taken up. These results revealed thatthe liposome encapsulating BCG-CWS is efficiently taken up into theMBT-2 cells.

(3) FACS Assay

A liposome encapsulating BCG-CWS was taken up into MBT-2 cells by themethod described in Example 3(2), followed by the washing of the cells.However, the incubation time after the addition of the liposome was setto 1 hour instead of 2 hours. Subsequently, 1 mL of an FCS-supplementedRPMI medium was added to the cells, which were then incubated for 1 hourunder the same conditions as above and used as a sample group. Also, thesame number of MBT-2 cells thereas in which no liposome encapsulatingBCG-CWS was taken up was used as a control group. The fluorescenceintensity of NBD and the number of cells were measured for the cells ofthe sample group and the control group using flow cytometry(FACSCalibur; Nippon Becton Dickinson Company Ltd.). The overallfluorescence intensity of the sample group was subjected to asignificance test (unpaired t-test) vs. the overall fluorescenceintensity of the control group. The overall fluorescence intensity ofeach group is shown in the left diagram of FIG. 10, and the relationshipbetween the fluorescence intensity of each cell and the number of cellsis shown in the right diagram of FIG. 10.

As shown in the left diagram of FIG. 10, the overall fluorescenceintensity of the sample group was significantly larger than the overallfluorescence intensity of the control group. As shown in the rightdiagram of FIG. 10, a large number of cells having large fluorescenceintensity were detected in the sample group compared with the controlgroup. The number of cells having large fluorescence intensity ascompared with the cells of the control group, among the cells of thesample group, was converted to a percentage and was consequently96.5±2.2%. These results demonstrated that a large number of cells inthe sample group took up the liposome encapsulating BCG-CWS.

These results of Examples 3(1) and 3(2) demonstrated that the lipidmembrane structure which has a particle size that permits filtrationsterilization, comprises a lipid bound with a peptide consisting of aplurality of arginine residues as a constituent lipid, and encapsulatesa bacterial cell component having dispersibility in a non-polar solventis efficiently taken up into cancer cells.

Example 4 Anticancer Effect (1) Effect on Transplanted Cancer Cell [1-1]Tumor Volume

Eight-week-old female C3H/HeN mice (Japan SLC, Inc.) were divided into 6groups (A to F) each involving 4 to 6 mice. MBT-2 cells were cultured at37° C. in a 5% (v/v) CO₂ atmosphere using an FCS-supplemented RPMImedium. Then, after removal of the medium, 3.5×10⁶ cells were suspendedin PBS to prepare a cell suspension. The cell suspension and eachliposome given below were placed in a microtube and mixed to obtain atransplant solution. The transplant solution was subcutaneously injectedto the right abdomens of the mice in each group using a 26 G needle anda tuberculin syringe to transplant the MBT-2 cells to the mice. Then,the mice in each group were reared for 25 days. The rearing was carriedout under conditions of standard temperature and humidity and 12-hourlight/12-hour dark cycles. In this period, the mice freely took feed anddrinking water. On days 14, 19, 21, and 25 after the transplantation,one to several mice were randomly selected from each group, and themajor axis and the minor axis of a tumor at the transplantation sitewere measured using a vernier caliper. In this context, the tumor iseasily distinguishable by visual observation as a raised cell mass.Subsequently, on the basis of the results of measuring the major axisand the minor axis, the tumor volume was calculated according to thefollowing expression 4:

Tumor volume (mm³)=0.52×Major axis×(Minor axis)².  Expression 4;

The tumor volumes of the groups C to F on day 25 after thetransplantation were subjected to a significance test (two-way repeatedmeasures ANOVA, Dunnett's method) vs. the groups A and B. The resultsare shown in FIG. 11.

“Liposome Mixed with Cell Suspension”

Group A (n=5): (cell suspension alone)

Group B (n=5): empty liposome of Example 1(1)[1-1] (amount of lipid:2.56 mg)

Group C (n=5): liposome encapsulating BCG-CWS of Example 1(1)[1-3](amount of BCG-CWS: 0.3 mg, amount of lipid: 2.56 mg)

Group D (n=4): liposome encapsulating BCG-CWS of Example 1(1)[1-3](amount of BCG-CWS: 0.1 mg, amount of lipid: 0.85 mg)

Group E (n=6): liposome containing BCG-CWS in lipid membrane of Example1(4) (amount of BCG-CWS: 0.3 mg, amount of lipid: 2.56 mg)

Group F (n=4): liposome containing BCG-CWS in lipid membrane of Example1(4) (amount of BCG-CWS: 0.1 mg, amount of lipid: 0.85 mg)

As shown in FIG. 11, the tumor volume on day 25 after thetransplantation tended to be small in the groups D and F compared withthe groups A and B and was significantly small in the groups C and E.These results revealed that the administration of the liposomeencapsulating BCG-CWS in addition to the transplantation of MBT-2 cellssuppresses the engraftment or growth of the transplanted MBT-2 cells.

[1-2] Survival Rate

Eight-week-old female C3H/HeN mice (Japan SLC, Inc.) were divided into 2groups (an administration group of the liposome encapsulating BCG-CWSand an administration group of the empty liposome) each involving 10mice. MBT-2 cells were transplanted to the mice in each group by themethod described in Example 4(1)[1-1]. The mice were reared forapproximately 60 days, and the number of surviving mice was counted.However, each liposome mixed with the cell suspension for the transplantsolution was as described below. The number of surviving mice in theadministration group of the liposome encapsulating BCG-CWS was subjectedto a significance test (logrank test) vs. the administration group ofthe empty liposome. The results are shown in FIG. 12. As shown in FIG.12, the number of surviving mice in the administration group of theliposome encapsulating BCG-CWS was significantly large as compared withthe administration group of the empty liposome. These results revealedthat the administration of the liposome encapsulating BCG-CWS inaddition to the transplantation of MBT-2 cells elevates the survivalrate.

“Liposome Mixed with Cell Suspension”

Administration group of the liposome encapsulating BCG-CWS: liposomeencapsulating BCG-CWS of Example 1(1)[1-3] (amount of BCG-CWS: 0.3 mg,amount of lipid: 2.56 mg)

Administration group of the empty liposome: empty liposome of Example1(1)[1-1] (amount of lipid: 2.56 mg)

[1-3] Area of Tumor Tissue and the Number of Leukocytes

MBT-2 cells were cultured at 37° C. for 1 hour in a 5% (v/v) CO₂atmosphere using an PCS-supplemented RPMI medium supplemented with theliposome encapsulating BCG-CWS of Example 1(1)[1-3] (amount of BCG-CWS:0.1 mg, amount of lipid: 0.85 mg) with respect to 1.0×10⁶ cells. Then,the cells were washed with PBS containing 20 U/mL heparin to remove aliposome that was not taken up into the cells. The MBT-2 cells in whichthe liposome encapsulating BCG-CWS was taken up were obtained by thistreatment and used as lipo-incorporated cells.

Four 8-week-old female C3H/HeN mice (Japan SLC, Inc.) were prepared anddesignated as mice a to d, respectively. As described below, 1.0×10⁶MBT-2 cells or lipo-incorporated cells were transplanted to each mouse,and each liposome was administered thereto. The mice were reared for 10days. The culture and transplantation of the MBT-2 cells, theadministration of the liposome, and the culture of the mice were carriedout in the same way as the methods described in Example 4(1)[1-1]. Then,tumor tissues were collected from each mouse and embedded in paraffinaccording to a routine method. Then, sections of approximately 3 μmthickness were prepared and subjected to hematoxylin-eosin staining andGiemsa staining. The images of the hematoxylin-eosin-stained sectionswere analyzed using image analysis software NIH Image Ver. 1.44p (WayneRasband, National Institute of Health, USA) to measure the total area oftumor tissues, the area of viable tumor tissues, the area of dead tumortissues, and the ratio of dead cells (%). Also, the numbers of variousleukocytes in the tumor tissues were measured by observation in the highpower field (HPF).

Mouse a: subcutaneous injection of a transplant solution of the MBT-2cells mixed with the empty liposome of Example 1(1)[1-1] (amount oflipid: 0.85 mg).

Mouse b: subcutaneous injection of a transplant solution of the MBT-2cells mixed with the liposome encapsulating BCG-CWS of Example 1(1)[1-3](amount of BCG-CWS: 0.1 mg, amount of lipid: 0.85 mg).

Mouse c: subcutaneous injection of a cell suspension containing thelipo-incorporated cells.

Mouse d: subcutaneous injection of a cell suspension containing theMBT-2 cells and PBS containing the liposome encapsulating BCG-CWS ofExample 1(1)[1-3] (amount of BCG-CWS: 0.1 mg, amount of lipid: 0.85 mg)at sites distant from each other.

The results of measuring the total area of tumor tissues, the area ofviable tumor tissues, the area of dead tumor tissues, and the ratio ofdead cells (%) are shown in FIG. 13. As shown in FIG. 13, in the mice bto d compared with the mouse a, the total area of tumor tissues and thearea of viable tumor tissues were remarkably small; the area of deadtumor tissues was at almost the same level, albeit small; and the ratioof dead cells was large. These results revealed that the administrationof the liposome encapsulating BCG-CWS in addition to the transplantationof MBT-2 cells suppresses the enlargement of tumor tissues derived fromthe transplanted MBT-2 cells and maintains or promotes the cell death inthe tumor tissues.

In the mice b and c compared even with the mouse d, the total area oftumor tissues and the area of viable tumor tissues were small, and theratio of dead cells was remarkably large. These results revealed thatthe transplantation of the mixture of the liposome encapsulating BCG-CWSwith the MBT-2 cells or the transplantation of the MBT-2 cells in whichthe liposome encapsulating BCG-CWS was taken up was more effective forsuppressing the enlargement of tumor tissues or for maintaining orpromoting the cell death in the tumor tissues.

The results of measuring the numbers and ratios of various leukocytes inthe tumor tissues are shown in FIG. 14. In the lower photographs of FIG.14, the cells having a round nucleus with dark color are lymphocytes. Asshown in FIG. 14, the mice b to d had large numbers of macrophages,lymphocytes, neutrophils, eosinophils, and mast cells and a large totalnumber of leukocytes as compared with the mouse a. As for the rate ofincrease in various leukocytes, lymphocytes were particularly increasedin the mice b to d compared with the mouse a. These results revealedthat the administration of the liposome encapsulating BCG-CWS inaddition to the transplantation of MBT-2 cells promotes the infiltrationof immunocytes, particularly, the infiltration of lymphocytes, intotumor tissues derived from the transplanted MBT-2 cells.

The mice b and c compared even with the mouse d had large numbers oflymphocytes, neutrophils, eosinophils, and mast cells and a large totalnumber of leukocytes in the tumor tissues and exhibited a large rate ofincrease in lymphocytes. These results revealed that the transplantationof the mixture of the liposome encapsulating BCG-CWS with the MBT-2cells or the transplantation of the MBT-2 cells in which the liposomeencapsulating BCG-CWS was taken up was more effective for infiltratingimmunocytes into the tumor tissues.

(2) Effect on Bladder Cancer Rat Model

Fifty four 6-week-old male F344/DuCrlCrlj rats (Charles RiverLaboratories Japan, Inc.) were prepared. These rats were reared for 8weeks while freely taking drinking water containing 0.05% of acarcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine and feed containing 5%of a cancer promoter sodium ascorbate so that bladder cancer was inducedtherein to prepare bladder cancer rat models. The bladder cancer ratmodels were divided into 6 groups (G to L) each involving 9 rats andreared while intravesically given a total of 8 doses (at 1-weekintervals) of a phosphate buffer solution containing each liposome givenbelow. For the intravesical administration, each rat was placed supineunder light ether anesthesia and alloyed to urinate by compression.Then, a cannula catheter attached with 24 G×¾ inch indwelling needle wasinserted through the external urethral opening and used in theadministration. In the rearing period during the liposome dosing period,the rats freely took usual feed and drinking water and were reared underconditions of standard temperature and humidity and 12-hourlight/12-hour dark cycles.

“Administered Liposome (Single Dose is Shown in the Parentheses <>)”

Group G: (non-administered)

Group H: empty liposome of Example 1(1)[1-1] <amount of lipid (in termsof the amount of BCG-CWS): 0.1 mg/1 mL×body weight of each ratindividual (kg)>

Group I: liposome encapsulating BCG-CWS of Example 1(1)[1-3] <amount ofBCG-CWS: 0.1 mg/1 mL×body weight of each rat individual (kg)>

Group J: liposome encapsulating BCG-CWS of Example 1(1)[1-3] <amount ofBCG-CWS: 0.03 mg/1 mL×body weight of each rat individual (kg)>

Group K: liposome containing BCG-CWS in a lipid membrane of Example 1(4)<amount of BCG-CWS: 0.1 mg/1 mL×body weight of each rat individual (kg)>

Group L: liposome containing BCG-CWS in a lipid membrane of Example 1(4)<amount of BCG-CWS: 0.03 mg/1 mL×body weight of each rat individual(kg)>

Each rat was reared for 1 week after the final administration and thendissected to excise its bladder, which was fixed by dipping 10% neutralbuffered formalin. The bladder thus fixed was divided into sagittalsections, and the weight was measured using an electronic balance. Inthis context, the bladder weight is considered to get larger with theprogression of bladder cancer. Then, the bladder was divided into 4portions and embedded in paraffin to prepare sections of approximately 3μm in thickness, followed by hematoxylin-eosin staining. The stainedsections were observed to count the number of epithelial lesions(tumors). The length of the bladder basement membrane was measured byimage analysis. On the basis of the measurement results, the number ofepithelial lesions (tumors) per 10 cm of the bladder basement membranewas calculated. Subsequently, the bladder weights and the numbers ofepithelial lesions (tumors) per 10 cm of the bladder basement membranein the groups I to L were subjected to a significance test (one-wayanalysis of variance, Dunnett's method) vs. the groups G and H. Thetumor volumes of the groups G to J were also measured by the methoddescribed in Example 4(1)[1-1]. The bladder was excised and cleaved intotwo. Digital photographs were taken on the mucosa side. Then, binarizedimages of tumor lesions were prepared to measure the number of lesionsand the tumor volume.

The tumor volumes of the groups G, I, and J were subjected to asignificance test (one-way analysis of variance, Dunnett's method) vs.the group H. The bladder sections of the groups G to J are shown in FIG.15. The bladder weights and the numbers of epithelial lesions (tumors)per 10 cm of the bladder basement membrane in the groups G to L and theresults of measuring the tumor volumes of the groups G to J are shown inFIG. 16.

As shown in the left diagram of FIG. 16, the bladder weight was smallestin the groups I and J among the groups G to L. The bladder weight wassignificantly small in the groups I to K compared with the group H.These results revealed that the administration of the liposomeencapsulating BCG-CWS to a bladder cancer rat model decreases thebladder weight or prevents increase in the bladder weight. As shown inFIG. 15, the number of epithelial lesions (tumors) in the bladder wasremarkably small in the groups I and J compared with the groups G and H.As shown in the central diagram of FIG. 16, the number of epitheliallesions (tumors) per 10 cm of the bladder basement membrane wassignificantly small in all of the groups I to L compared with the groupH. The number of epithelial lesions (tumors) per 10 cm of the bladderbasement membrane was significantly small in the groups J to L comparedeven with the group G. These results revealed that the administration ofthe liposome encapsulating BCG-CWS to a bladder cancer rat modeldecreases the number of epithelial lesions (tumors) or prevents increasein the number of epithelial lesions (tumors). As shown in the rightdiagram of FIG. 16, the tumor volume was small in the groups I and Jcompared with the groups G and H. The tumor volume was significantlysmall particularly in the group I compared with the group H. Theseresults revealed that the administration of the liposome encapsulatingBCG-CWS to a bladder cancer rat model decreases the tumor volume orprevents increase in the tumor volume.

These results of Examples 4(1)[1-1] to 4(2) demonstrated that the lipidmembrane structure which has a particle size that permits filtrationsterilization, comprises a lipid bound with a peptide consisting of aplurality of arginine residues as a constituent lipid, and encapsulatesa bacterial cell component having dispersibility in a non-polar solventcan attain the treatment of cancers or the inhibition of the progressionof cancers. These results also demonstrated that the lipid membranestructure which has a particle size that permits filtrationsterilization, comprises a lipid bound with a peptide consisting of aplurality of arginine residues as a constituent lipid, and encapsulatesa bacterial cell component having dispersibility in a non-polar solventexerts higher anticancer effect through direct administration to cancercells.

Example 5 Immunostimulatory Activity (1) Isolation of Naive CD4-PositiveT Cell

Blood was collected from each of 4 healthy test subjects, and peripheralblood mononuclear cells were isolated using Ficoll-Paque (Pharmacia &Upjohn Company LLC). Subsequently, the cells were reacted with anFITC-labeled anti-CD8/CD45RO antibody to label naive CD4-positive Tcells with FITC. Next, the FITC-labeled cells were isolated usinganti-FITC magnetic beads (Miltenyi Biotec) and Auto-MACS cell sorter(Miltenyi Biotec) to obtain naive CD4-positive T cells.

(2) Addition of Liposome

A Mycobacterium Bovis bacillus Calmette-Guerin cell-wall (BCG-CW)fraction suspended in PBS, the empty liposome of Example 1(1)[1-1], andthe liposome encapsulating BCG-CWS of Example 1(1)[1-3] were each addedto a medium for the naive CD4-positive T cells of Example 5(1) and usedas a positive control group, a negative control group, and a test group,respectively. The amount of BCG-CW or BCG-CWS added was 1, 3, 10 and 30μg/mL as final concentrations. Non-supplemented naive CD4-positive Tcells were used as a reference group. Subsequently, these cell groupswere each cultured for 1 week in a CO₂ incubator of 37° C. using amedium for induction of differentiation into Th1 cells or Th2 cells. Inthis 1-week culture period, the first 2 days were directed to culture ineach medium supplemented with 20 μg/mL anti-CD3 antibody, and theremaining 5 days were directed to culture in each medium unsupplementedwith the anti-CD3 antibody. The medium for induction of differentiationinto Th1 cells used was an RPMI1640 medium containing 10% serum andcontaining 50 U/mL IL-2 (Shionogi & Co., Ltd.), 1 ng/mL IL-12 (R & DSystems, Inc.), and 5 μg/mL anti-IL-4 antibody (BD Biosciences). Themedium for induction of differentiation into Th2 cells used was anRPMI1640 medium containing 10% serum and containing 50 U/mL IL-2(Shionogi & Co., Ltd.), 1 ng/mL IL-4 (R & D Systems, Inc.), and 5 μg/mLanti-IFN-γ antibody (BD Biosciences). Then, the cells were reacted withan anti-IFN-γ antibody and an anti-IL-4 antibody, and IFN-γ-producingcells and IL-4-producing cells were detected by flow cytometry(FACSCalibur; Nippon Becton Dickinson Company Ltd.). The results aboutone person randomly selected from the 4 test subjects are shown in FIG.17.

As shown in the upper diagram of FIG. 17, in the case of using themedium for induction of differentiation into Th1 cells (under theenvironment to induce differentiation into Th1), the number of cellsproducing IFN-γ and producing no IL-4 (Th1 cells) was large in the testgroup compared with the negative control group, regardless of the amountof BCG-CW or BCG-CWS added. Particularly, when the amount of BCG-CW orBCG-CWS added was 30 μg/mL, the number of Th1 cells in the test groupwas larger than that in the negative control group and the referencegroup and was equivalent to that in the positive control group. Theseresults revealed that the liposome encapsulating BCG-CWS promotes thedifferentiation of naive CD4-positive T cells into Th1 cells under theenvironment to induce differentiation into Th1.

On the other hand, as shown in the lower diagram of FIG. 17, in the caseof using the medium for induction of differentiation into Th2 cells(under the environment to induce differentiation into Th2), the numberof cells producing no IFN-γ and producing IL-4 (Th2 cells) in the testgroup was smaller than that in the negative control group and thereference group and was equivalent to that in the positive controlgroup, regardless of the amount of BCG-CW or BCG-CWS added. When theamount of BCG-CW or BCG-CWS added was 3, 10, and 30 μg/mL, the number ofTh1 cells in the test group was larger than that in the negative controlgroup and the reference group and was equivalent to that in the positivecontrol group. These results revealed that the liposome encapsulatingBCG-CWS promotes the differentiation of naive CD4-positive T cells intoTh1 cells and suppresses the differentiation thereof into Th2 cells evenunder the environment to induce differentiation into Th2.

Next, the rate at which the naive CD4-positive T cells were convertedinto IFN-γ-producing cells or IL-4-producing cells (rate of conversion;%) was calculated on the basis of the flow cytometry measurementresults. The rate of conversion was calculated as the ratio ofIFN-γ-producing cells or IL-4-producing cells in each group when theratio of IFN-γ-producing cells or IL-4-producing cells in the referencegroup was defined as 100%. Subsequently, a mean of the results about the4 test subjects and standard deviation were determined as to the rate ofconversion. The rate of conversion of the test group was subjected to asignificance test (unpaired t-test) vs. the negative control group. Theresults are shown in FIG. 18.

As shown in FIG. 18, the rate of conversion into IFN-γ-producing cellsin the test group tended to be large as compared with the negativecontrol group both under the environment to induce differentiation intoTh1 and under the environment to induce differentiation into Th2.Particularly, when the amount of BCG-CW or BCG-CWS added was 30 μg/mLunder the environment to induce differentiation into Th2, the rate ofconversion into IFN-γ-producing cells in the test group wassignificantly large as compared with the negative control group. Whenthe amount of BCG-CW or BCG-CWS added was 3, 10, and 30 μg/mL, the rateof conversion into IL-4-producing cells in the test group under theenvironment to induce differentiation into Th2 was significantly smallas compared with the negative control group. Also when the amount ofBCG-CW or BCG-CWS added was 1 μg/mL, this rate of conversion tended tobe small as compared with the negative control group. These resultsrevealed that the liposome encapsulating BCG-CWS promotes thedifferentiation of naive CD4-positive T cells into Th1 cells both underthe environment to induce differentiation into Th1 and under theenvironment to induce differentiation into Th2. These results alsorevealed that the liposome encapsulating BCG-CWS suppresses thedifferentiation of naive CD4-positive T cells into Th2 cells under theenvironment to induce differentiation into Th2.

These results of Example 5(2) demonstrated that the lipid membranestructure which has a particle size that permits filtrationsterilization, comprises a lipid bound with a peptide consisting of aplurality of arginine residues as a constituent lipid, and encapsulatesa bacterial cell component having dispersibility in a non-polar solventacts on immunocytes to activate cellular immunity.

1. A lipid membrane structure having a particle size that permitsfiltration sterilization, comprising a lipid bound with a peptideconsisting of a plurality of arginine residues as a constituent lipidand encapsulating a bacterial cell component having dispersibility in anon-polar solvent.
 2. The lipid membrane structure according to claim 1,wherein the bacterial cell component is cell-wall fraction (CW) orcell-wall skeleton fraction (CWS) of one or more bacteria selected fromthe group consisting of a bacterium of the genus Mycobacterium, abacterium of the genus Nocardia, a bacterium of the genusCorynebacterium, and a bacterium of the genus Rhodococcus.
 3. The lipidmembrane structure according to claim 2, wherein the bacterium of thegenus Mycobacterium is Mycobacterium Bovis bacillus Calmette-Guerin(BCG).
 4. The lipid membrane structure according to claim 1, wherein thelipid membrane structure having a particle size that permits filtrationsterilization is a lipid membrane structure having a particle size of180 nm or smaller.
 5. A pharmaceutical composition comprising a lipidmembrane structure according to claim
 1. 6. The pharmaceuticalcomposition according to claim 5, wherein the pharmaceutical compositionis a therapeutic agent for bladder cancer and/or an agent inhibiting theprogression thereof.
 7. A method for producing a lipid membranestructure having a particle size that permits filtration sterilizationand encapsulating a substance of interest having dispersibility in anon-polar solvent, comprising the following steps (i) to (iv): (i)preparing a polar solvent solution containing a lipid membrane structureencapsulating no substance of interest; (ii) preparing a non-polarsolvent solution in which the substance of interest dispersed; (iii)mixing the polar solvent solution containing a lipid membrane structureencapsulating no substance of interest with the non-polar solventsolution in which the substance of interest dispersed to prepare anoil-in-water emulsion; and (iv) distilling off the non-polar solventunder reduced pressure from the oil-in-water emulsion.
 8. The methodaccording to claim 7, wherein the lipid membrane structure encapsulatingno substance of interest is a lipid membrane structure comprising alipid bound with a peptide consisting of a plurality of arginineresidues as a constituent lipid and encapsulating no substance ofinterest.
 9. The method according to claim 7, wherein the substance ofinterest is a bacterial cell component.
 10. The method according toclaim 9, wherein the bacterial cell component is cell-wall fraction (CW)or cell-wall skeleton fraction (CWS) of one or more bacteria selectedfrom the group consisting of a bacterium of the genus Mycobacterium, abacterium of the genus Nocardia, a bacterium of the genusCorynebacterium, and a bacterium of the genus Rhodococcus.
 11. Themethod according to claim 10, wherein the bacterium of the genusMycobacterium is Mycobacterium Bovis bacillus Calmette-Guerin (BCG). 12.The method according to claim 7, wherein the particle size that permitsfiltration sterilization is a particle size of 180 nm or smaller.